The long-term goal is to understand the physiological and pathological roles of Rab GTPases in cells and organisms. This project is focused on two closely related Rab GTPases (Rab22 and Rab5) that are localized in early endosomes and regulate endosomal sorting and endocytosis. Specifically the hypothesis that Rab22 is evolved from Rab5 to accommodate the need for regulation of specialized cell differentiation processes in higher eukaryotes, such as nerve growth factor (NGF)-mediated neuronal differentiation and survival, will be investigated. Malfunction in NGF signal transduction plays an important role in neurodegenerative diseases such as Alzheimer's disease and Down syndrome. We found that inhibition of Rab22 function via RNAi or dominant negative mutants dramatically reduces NGF-induced VGF expression and neurite outgrowth in PC12 cells, in contrast to the stimulatory effect of dominant negative Rab5 mutants. Furthermore, the activated NGF receptor (pTrkA) is endocytosed and localized to Rab22-containing endosomes. The preliminary data suggest an important role for Rab22 in the biogenesis and function of NGF-pTrkA signaling endosomes that promote cell differentiation and survival. This project contains three specific aims to investigate the mechanism of Rab22 in NGF signal transduction and cell differentiation in PC12 cells as well as neurons and to determine the structural differences that distinguish Rab22 from Rab5 in terms of promoting NGF signal transduction and cell differentiation. Aim 1 will test if Rab22 knockdown via RNAi may abrogate the formation of NGF-pTrkA signaling endosomes and consequently diminish the level and duration of pTrkA in PC12 cells and the activities of downstream effectors (Rap1 and ERKs). Furthermore, the experiments will determine Rab22- specific effectors responsible for the function in NGF-induced activation of ERKs, VGF expression, and neurite outgrowth. Aim 2 will test if Rab22 knockdown may abrogate the retrograde trafficking and signaling of NGF- pTrkA signaling endosomes in dorsal root ganglia (DRG) neurons and lead to apoptosis. Aim 3 will test if and what mutations in Rab5 may give rise to Rab22 characteristics in terms of specificity in interactions with effectors and function in NGF signaling and cell differentiation. In summary, this project will contribute to understanding the evolution of the Rab GTPase family in terms of structural and functional specificity of Rab22 and Rab5. Furthermore, it develops a novel concept that the early endosome-associated Rab22 is critical for the biogenesis and function of signaling endosomes that promote NGF signaling and neuron differentiation, which will contribute to understanding the mechanism of neurodegenerative diseases and developing potential therapeutics.